STAGE SPECIFIC GENES IN LEISHMANIA ENRIETTII

利什曼原虫 ENRIETTII 阶段特异性基因

• 批准号: 2063181
• 负责人: Scott M Landfear
• 金额: $ 13.77万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2063181
• 关键词: Leishmania; autoradiography; developmental genetics; electroporation; environmental adaptation; gene expression; genetic promoter element; genetic regulation; genetic transcription; guinea pigs; histones; immunoelectron microscopy; immunofluorescence technique; laboratory rabbit; life cycle; membrane proteins; microorganism genetics; molecular cloning; molecular genetics; protein sequence; protein structure function; radiotracer; restriction mapping; scintillation spectrometry; southern blotting; transfection; transport proteins; western blottings

Leishmania are parasitic protozoa that cause a major, life-threatening disease which afflicts millions of people living in tropical regions of the globe. The major objective of this proposal is to study several genes in Leishmania parasites whose expression is restricted to the stage of the life cycle that inhabits the insect vector. These genes code for membrane transport proteins which are apparently involved in the parasite's adaption to the environment of the insect gut, probably by allowing the parasite to utilize nutrients which are available in the insect but not in the mammalian host. One of these genes, designated Pro-1, codes for a protein related in sequence known glucose transport proteins from mammals, yeast and bacteria and which probably transports either glucose or another sugar. The ligand that is transported by the Pro-1 protein will be determined by transfecting Leishmania parasites with this cloned gene under conditions which will allow accumulation of many copies of the transformed gene. These transfected parasites will then be assayed for increased ability to transport various radiolabeled sugar ligands, compared to untransfected parasites. In addition, the location of the Pro-1 protein within the parasite will be investigated by immunoelectron microscopy to determine whether it is located in the plasma membrane and whether it is uniformly distributed or localized to a specific region of the membrane. Finally, the transcription of the Pro-1 gene will be investigated to determine where its promoter is located. Understanding transcription of the Pro-1 gene may help reveal how this gene is regulated during the parasite life cycle, being expressed in the insect from parasites but not in the form that infects the mammalian host. Two other genes that code for related membrane transport proteins, designated D1 and D2, will also be studied in detail. Complete genomic copies of D1 and D2 will be isolated and sequenced to determine the deduced amino acid sequence of each protein. The ligands which are transported by D1 and D2 will be determined, either by transfecting parasites with many copies of each gene and assaying for increased transport activity or be disrupting each gene and assaying for loss of specific transport function. Together these studies will advance our understanding of how genes are regulated during the parasite life cycle, allowing the parasite to adapt both its host and insect vector, and of how membrane transport proteins function to allow the parasite to survive in its changing environment.

利什曼原虫是一种寄生原生动物，可引起严重的、危及生命的疾病 这种疾病困扰着生活在热带地区的数百万人 地球。 该提案的主要目标是研究 表达仅限于阶段的利什曼原虫寄生虫 栖息于昆虫媒介的生命周期。 这些基因编码膜 显然参与寄生虫适应的转运蛋白 昆虫肠道的环境，可能是通过允许寄生虫 利用昆虫体内可利用但昆虫体内无法利用的营养物质 哺乳动物宿主。 其中一个基因被称为 Pro-1，编码与 对来自哺乳动物、酵母和细菌的已知葡萄糖转运蛋白进行测序 它可能运输葡萄糖或另一种糖。 配体 由 Pro-1 蛋白转运的值将由下式确定 在一定条件下用该克隆基因转染利什曼原虫 这将允许转化基因的许多拷贝的积累。 然后将检测这些转染的寄生虫的增强能力 与未转染相比，转运各种放射性标记的糖配体 寄生虫。 此外，Pro-1 蛋白在 将通过免疫电子显微镜研究寄生虫以确定 是否位于质膜内以及是否均匀 分布或定位于膜的特定区域。 最后， 将研究 Pro-1 基因的转录以确定其位置 它的启动子位于。 了解 Pro-1 基因的转录 可能有助于揭示该基因在寄生虫生命周期中是如何受到调节的， 在昆虫中通过寄生虫表达，但不以以下形式表达 感染哺乳动物宿主。 另外两个编码相关膜转运蛋白的基因， 指定为D1和D2，也将进行详细研究。 完整基因组 D1 和 D2 的副本将被分离并测序以确定推论 每种蛋白质的氨基酸序列。 转运的配体 D1 和 D2 将通过用许多转染寄生虫来确定 每个基因的拷贝并测定增加的运输活性或 破坏每个基因并检测特定转运功能的丧失。 这些研究将共同​​推进我们对基因如何运作的理解 在寄生虫生命周期中受到调节，使寄生虫能够适应 它的宿主和昆虫载体，以及膜转运蛋白的方式 功能使寄生虫能够在不断变化的环境中生存。